Fig 1.
Chemical structures thia/isothiacalothrixin B analogues.
The calothrixin B (CAB 1) is the naturally occurring anti-cancer alkaloid from the cyanobacteria Calothrix. The carbazole nitrogen group (-NH) was replaced with sulphur atom to result in thiacalothrixin B (SCAB 1) which upon isomerization results in isothiacalothrixin B (SCAB 4). The substitution with fluorine atom on the E-ring or A-ring of isothiacalothrixin B results in SCAB 6 and 11, respectively.
Fig 2.
Isothiacalothrixins caused potent growth inhibition of cancer cell lines.
Dose-response graphs of calothrixin B analogues assayed in a panel of nine cancer cell lines. Overlays of the percent growth of all nine cell lines following a 48 hours treatment with varying doses of calothrixin B (A), thiacalothrixin B (B), isothiacalothrixin B (C), 3-Fluoro isothiacalothrixin (D), 11-Fluoro isothiacalothrixin (E). HeLa (solid green circle), SiHa (solid blue triangle), MCF7 (solid cyan inverted triangle), NIC-H460 (solid magenta diamond), MDA-MB231 (solid yellow left-sided triangle), U251 (solid yellow right-sided triangle), HCT-116 (solid navy hexagon).
Table 1.
In vitro cytotoxicity data for calothrixin B and its thia analogues against seven human tumour cell lines.
Fig 3.
Isothiacalothrixins severely affected the clonogenicity of HCT116 and NCI-H460 cells.
The effects of calothrixin B, thiacalothrixin B and isothiacalothrixin analogues on the clonogenic growth of colon adenocarcinoma HCT116 and NCI-H460 cell lines. Cells were seeded in a six-well plate and after over-night adherence, treated with different concentrations (0.1–5 μM) of calothrixin B, thiacalothrixin B and isothiacalothrixins (SCAB 4, 6 and 10), for 48 hours. After drug treatment, the cells were washed with Dulbecco’s phosphate buffered saline and let grow up to 14 days in drug-free medium. Cell colonies were stained with crystal violet and photographed.
Fig 4.
Isothiacalothrixin B (SCAB 4) analogues caused time- and dose-dependent arrest of cells in S and G2/M phases.
(A) Cell cycle analysis of human colon HCT116 cells treated with different concentrations (1, 2 and 5 μM) of the thiacalothrixins B (SCAB 1) or (0.5, 1 and 2 μM) isothiacalothrixins B (SCAB 4) for 24 and 48 hours. (B) The table shows the Mean ± SD percentage of cells in the G0/G1, S and G2/M phases of the cell cycle. Figures are representative of other two experiments.
Fig 5.
Isothiacalothrixin B (SCAB 4) but not thiacalothrixin B caused the arrest of cells in S and G2/M phases.
Cell cycle perturbation by thiacalothrixin B (SCAB 1) or isothiacalothrixin B (SCAB 4, SCAB 10) in the presence or absence of nocodazole. HCT116 cells were treated with 5 μM of thiacalothrixin B and its analogues for 20 hours or 3 hours pretreatment with 17 hours in the presence of nocodazole followed by propidium iodide staining. The population of cells in different phases of the cell cycle were analysed by flow cytometry. (B) The table shows the Mean ± SD percentage of cells in the G0/G1, S and G2/M phases of the cell cycle. Figures are representative of other two experiments.
Fig 6.
Thiacalothrixin B and its analogues cleaved plasmid DNA in a cell-free system.
Supercoiled plasmid DNA was incubated for 1 hour with 250 μM of thiacalothrixin B analogues, 200 μM of ferric chloride in the presence or absence of DTT (200 μM). Plasmid DNA was separated by agarose gel and stained with ethidium bromide.
Fig 7.
Thia analogues of calothrixin B analogue induced DNA strand breaks.
Single-cell gel electrophoresis data (comet assay) in HCT116 cells (A) treated for 48 hours with 5 μM thiacalothrixin B, SCAB 1 (B) or 5 μM isothiacalothrixin B, SCAB 4 (C). Olive tail moment (D) and mean percentage tail DNA (E) in HCT116 cells exposed to 5 μM of SCAB1 or SCAB4 for 48 hours. Values are represented as Mean ± standard deviation; *, P < 0.05. Experiments were performed in duplicate.
Table 2.
In silico calculations of free binding energy scores for the calothrixin B and thiacalothrixin B analogues ligands docked with octamer DNA.
Fig 8.
The isothiacalothrixin B analogues fit deep into the minor groove of DNA.
Best docking conformations of calothrixin B and its thia analogues with crystallographic DNA (PDB ID: 453D DNA) towards left and a two-dimensional representation of key structural moieties along with their base-pair interactions depicted on to the right.
Table 3.
Docking results of calothrixin B and its thia analogues with DNA (PDB ID: 453D).
Fig 9.
Calothrixin B or thiacalothrixin B but not isothiacalothrixins induced p21 with no upregulation of p53.
Western blot analysis of p53 and p21waf1/cip1 protein in HCT116 cells after treatment without or with 5 μM thiacalothrixin B analogues for 48 hours. The whole cell lysate was immunoblotted with p53 and p21 antibody, respectively. The same blot was re-probed with β-actin to confirm equal loading of each lane.
Fig 10.
Transcriptional activation of p21 by calothrixin B or thiacalothrixin B independent of p53.
HCT116 cells were transfected with the wild-type (p21P-luc2) or the truncated p21 (p21PΔp53-luc2) promoter region, exposed to different concentrations of calothrixin B or thicalothrixin B analogues for 48 hours, then harvested and assayed for luciferase activity. Histograms represented firefly luciferase activity in each sample and expressed as fold induction relative to controls for each reporter, of which the values were taken as 1. All the values are the average of triplicate samples from a typical experiment; bars, ±SE.
Fig 11.
Isothiacalothrixin B analogues induced apoptotic morphology.
HCT116 cells were stained with acridine orange/ethidium bromide after 48 hours of treatment with calothrixin B/ thiacalothrixin analogues. Cells were observed under fluorescence microscope (x100 magnification). Viable cells show green fluorescence. Necrotic and apoptotic cells show orange and yellow fluorescence. (A) Untreated HCT116 control cells. Cells were treated with 5 μM of calothrixin B, CAB 1 (B) 5 μM of thiacalothrixin B, SCAB 1 (C), 5 μM of isothiacalothrixin B SCAB 4 (D), 5 μM of 3-Fluoro isothiacalothrixin B, SCAB 6 (E), 5 μM of 11-fluoro isothiacalothrixin B, SCAB 10 (F). Cells were observed under fluorescence microscope (x400 magnification), (a) Untreated HCT116 control cells. Cells were treated with 5 μM of thiacalothrixin B, SCAB 1 (b), 5 μM of 11-Fluoro isothiacalothrixin B, SCAB 10 (c).
Fig 12.
Isothiacalothrixin B analogues induced DNA fragmentation.
HCT116 cells were treated with 5 uM of thiacalothrixin B (SCAB 1) or isothiacalothrixin analogues (SCAB 4, 10) The cells were processed as described under "Materials and Methods section". The isolated DNA was electrophoresed on TAE agarose gel for 1.5 h at 100 V. The DNA fragments were visualised by staining with ethidium bromide. Lane 1 = DNA from untreated HCT116 cells; lane 2 = DNA from thiacalothrixin B (SCAB 1) treated cells; lane 3 = DNA from isothiacalothrixin B (SCAB 4) treated cells; lane 4 = DNA from 11-Fluoro isothiacalothrixin B (SCAB 10) treated cells.
Fig 13.
The cell cycle effects of isothiacalothrixin B (SCAB 4) were irreversible.
(A) Cell cycle perturbation at 24, 48, 72 and 96 hours post-treatment for 4 hours with thiacalothrixin B (SCAB 1) or isothiacalothrixin B (SCAB 4). HCT116 cells were treated with 5 μM of thiacalothrixin B and isothiacalothrixin B for 4 h followed by washing with PBS, and collection of cells at 24, 48, 72 and 96 hours in the recovery period and staining with propidium iodide. The percent population of cells in different phases of the cell cycle (B) were analyzed by flow cytometry. The figure is representative of other two experiments.